1. Field of the Invention
This invention relates to electrophysiological testing of biological samples. In particular, this invention relates to electrophysiological testing of agonists, antagonists, modulators, and other molecular species to determine their effect upon ion channels, electrical potential of cell membrane, and electrical currents through cell membranes.
2. Discussion of the Art
Electrophysiological methods provide the best and, often, the only available approach for studying and testing responses of ion channels of cell membranes. Such channels, which control the flow of ions across cell membranes and regulate the electrical potential of cells, are critically important for the proper functioning of plant and animal cells. Well-known examples of this are found in the nervous, muscle, cardiovascular, endocrine, and immune systems. Understanding the actions of substances that regulate ion channels (e.g., neurotransmitters, hormones, alkaloids, toxins, alcohols, and anesthetics) and discovering novel therapeutics that act through ion channels are important enterprises that are dependent upon electrophysiological approaches and the testing of ion channels in biological membranes. However, conventional methods have been hampered by low throughput, even in facile models such as transfected Xenopus oocytes.
The simplest method of increasing the rate of data collection is to multiply the number of conventional electrophysiological testing stations and the personnel needed to operate them. However, such actions increase costs for equipment, floor space, overhead, and personnel. Because of the initial cost of a conventional workstation, the floor space required, and the omnipresent difficulties in recruiting and retaining qualified electrophysiologists, this approach is not ideal.
Some researchers have used a manifold system, wherein many tubes of solutions are brought into a single chamber, with the individual tubes being controlled by solenoid valves (which can operate under the control of a computer). See, for example, ValveBank8 Perfusion System, commercially available from AutoMate Scientific, Inc. (Oakland, Calif.). This approach allows automated experiments to be performed, but still requires tedious, manual priming operations to be performed for each compound at each concentration. In addition, the duration of a given experiment is dominated by the duration of flow channel cleaning and duration of receptor recovery, and not duration of data collection. Thus, the system is idle for most of the experiment, effectively wasting resources. Increasing throughput would require complete system replication.
The “OTC-20” instrument (ALA Scientific Instruments, Westbury, N.Y.) utilizes a 20-sample carousel and provides low dead volume with random access to reagents for a single Xenopus oocyte. This system employs a movable, closed oocyte flowcell having electrodes integrated therewith. This flowcell has a bottom orifice that allows it to be dipped into a Petri dish containing the reagents needed for the experiment. A rotating carousel allows random access to Petri dishes containing solutions of the compound being tested. However, the system is limited to one oocyte and only 20 test samples at a time. The crude method of random access prevents the reagent vessels common in the pharmaceutical industry from being used and severely limits the number of samples that can be tested in one operation. Loading of the oocytes into the flowcell is also difficult and not amenable to high throughput. This approach also suffers from the two difficulties previously discussed, namely the apparatus spends the majority of the experiment waiting for the oocyte to recover, and increases in throughput require multiple systems.
In a conventional method of data analysis, data from each response is stored as a coded file, the relevant information existing as text tags in the file (see, for example, Clampex, available from Axon Instruments, incorporated herein by reference). To construct a dose response curve, these files must be analyzed individually by means of a separate software program (see, for example, Clamp-Fit, available from Axon Instruments, incorporated herein by reference). The results of each of these separate analyses are normalized to similar measurements of the responses from reference (control) agonist. In general, a simple normalization scheme is used because of the tedious nature of the operation. The responses at the required doses are averaged for an individual test subject and a table of results is constructed. A series of tables is constructed for the same test material on a varying number of test subjects. This series of tables is then imported into a curve-fitting package (see, for example, Prism, available from GraphPad, incorporated herein by reference), where the appropriate parameters are extracted. These parameters are used to create another series of table entries to be exported into a database for long-term storage and integration with other data. All of these steps are manual “cut and paste” operations, employing several software products. These steps are not only very time-consuming, but also susceptible to error due to their manual and highly repetitive nature.
Thus, there is a clear need for methods and devices to augment throughput in electrophysiological data acquisition and analysis, thereby increasing productivity.